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Literature summary extracted from

  • Zou, X.W.; Liu, Y.C.; Hsu, N.S.; Huang, C.J.; Lyu, S.Y.; Chan, H.C.; Chang, C.Y.; Yeh, H.W.; Lin, K.H.; Wu, C.J.; Tsai, M.D.; Li, T.L.
    Structure and mechanism of a nonhaem-iron SAM-dependent C-methyltransferase and its engineering to a hydratase and an O-methyltransferase (2014), Acta Crystallogr. Sect. D, 70, 1549-1560.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.281 gene mppJ from the mannopeptimycin-biosynthetic gene cluster, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His6-tagged enzyme Streptomyces hygroscopicus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.281 purified recombinant His6-tagged enzyme, in apoform or in complex with S-adenosyl-L-methionine and phenylpyruvate, hanging drop vapour diffusion technique, mixing of 0.001 ml of 24 mg/ml protein in 50 mM HEPES, pH 7.5, 100 mM CaCl2, with 0.001 ml of reservoir solution containing 16% PEG 3350, 200 mM NaI, 20°C, 10 days, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, molecular replacement. From the collected crystal diffraction data four additional structures of MppJ in complex with phenlpyruvate, 4-hydroxyphenylpyruvate, S-adenosyl-L-methionine and phenylpyruvate or S-adenosyl-L-homocysteine and methylphenylpyruvate, although crystals of the apo form remain unobtainable Streptomyces hygroscopicus

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.281 C319A site-directed mutagenesis, the mutant shows 39% reduced activity compared to the wild-type enzyme Streptomyces hygroscopicus
2.1.1.281 D244E site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme Streptomyces hygroscopicus
2.1.1.281 D244L site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme Streptomyces hygroscopicus
2.1.1.281 R127L/D244E site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme Streptomyces hygroscopicus
2.1.1.281 W99F the mutant shows altered stereochemistry in the reaction compared to the wild-type enzyme Streptomyces hygroscopicus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.281 Fe2+ required, binding structure, overview Streptomyces hygroscopicus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.1.1.281 38000
-
2 * 38000, recombinant His6-tagged enzyme, SDS-PAGE Streptomyces hygroscopicus
2.1.1.281 70200
-
recombinant His-tagged enzyme, gel filtration Streptomyces hygroscopicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate Streptomyces hygroscopicus phenylpyruvate binding structure analysis S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate Streptomyces hygroscopicus NRRL3085 phenylpyruvate binding structure analysis S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.281 Streptomyces hygroscopicus Q643C8 gene mppJ
-
2.1.1.281 Streptomyces hygroscopicus NRRL3085 Q643C8 gene mppJ
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.281 recombinant N-terminally His6-tagged enzyme by nickel affinity chromatography, ion-exchange chromatography, and ultrafiltration Streptomyces hygroscopicus

Reaction

EC Number Reaction Comment Organism Reaction ID
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate = S-adenosyl-L-homocysteine + (3S)-2-oxo-3-phenylbutanoate the enzyme methylates the benzylic C atom of phenylpyruvate to give 3-methylpyruvate, reaction mechanism, structure-function analysis, overview Streptomyces hygroscopicus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.281 additional information the enzyme has no nonheme oxygenase activity Streptomyces hygroscopicus ?
-
?
2.1.1.281 additional information the enzyme has no nonheme oxygenase activity Streptomyces hygroscopicus NRRL3085 ?
-
?
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate phenylpyruvate binding structure analysis Streptomyces hygroscopicus S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate Arg127 is electrostatically associated with phenylpyruvate, binding structure analysis, enzyme MppJ is an R-configuration-specific methyltransferase, ratios of 5:1 and 1:1 for (2S,3R)-betaMePhe versus (2S,3S)-betaMePhe Streptomyces hygroscopicus S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate phenylpyruvate binding structure analysis Streptomyces hygroscopicus NRRL3085 S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?
2.1.1.281 S-adenosyl-L-methionine + 3-phenylpyruvate Arg127 is electrostatically associated with phenylpyruvate, binding structure analysis, enzyme MppJ is an R-configuration-specific methyltransferase, ratios of 5:1 and 1:1 for (2S,3R)-betaMePhe versus (2S,3S)-betaMePhe Streptomyces hygroscopicus NRRL3085 S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate
-
?

Subunits

EC Number Subunits Comment Organism
2.1.1.281 homodimer 2 * 38000, recombinant His6-tagged enzyme, SDS-PAGE Streptomyces hygroscopicus

Synonyms

EC Number Synonyms Comment Organism
2.1.1.281 MppJ
-
Streptomyces hygroscopicus

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.281 S-adenosyl-L-methionine
-
Streptomyces hygroscopicus

General Information

EC Number General Information Comment Organism
2.1.1.281 evolution enzyme MppJ is structurally related to the MT protein family Streptomyces hygroscopicus
2.1.1.281 additional information the enzyme is composed of two domains: a dimerization domain (DD) and a methyltransfer domain (MT). The substrate-binding pocket is situated at the occlusion of the DD and MT domains, in which the iron centre is solvent-accessible Streptomyces hygroscopicus